This application responds to an RFA for approaches to new natural products and the new natural products themselves that would increase the biologically relevant molecular diversity in the NIH Molecular Libraries Roadmap and other screening initiatives. Two approaches form the application's two Specific Aims. Both emphasize a genetic starting point for discovering new natural products from bacterial sources. Specific Aim 1 focuses on Gammaproteobacteria, and Specific Aim 2 focuses on actinomycetes. Bacterial sources are emphasized because of their past contributions, recent studies that show their potential has been only poorly explored, their genetic accessibility, and their ability to be cultured and provide the multimilligram amounts of material needed for screening programs. The two aims not only use very different groups of bacteria, they also use different starting points, uncharacterized genomic DNA and genome sequences, to discover bioactive small molecules. Specific Aim 1. Discover biologically active small molecules through gene-to- molecule approaches. The first approach utilizes cosmid libraries from genomic DNA of Photorhabdus spp. and Xenorhabdus spp., heterologous expression in the metabolically compatible host Escherichia coli, and a functional assay for antibiotic activity, which serves as a surrogate assay for many other activities. Both genera, Photorhabdus and Xenorhabdus, are likely to produce large numbers of antibiotic molecules, and their close relationship to the heterologous host, E. coli, facilitates high levels of expression of the small molecules in this DNA library approach. Specific Aim 2. Develop sequence-based approaches to discovering natural products from 20 new actinomycete genomes. The largely function-based approach of Specific Aim 1 will be complemented by a sequence-based approach that leverages ongoing efforts to sequence and annotate the secondary biosynthetic pathways of 20 actinomycete strains in Specific Aim 2. This aim uses `knockout scanning'to identify small molecules associated with predicted gene clusters. For strains refractory to genetic manipulation, heterologous expression in appropriate hosts will be used. PUBLIC HEALTH RELEVANCE: High-throughput screening (HTS), the testing of large numbers of small molecules in biological assays, is the major tool for discovering new drugs, and the quality of the small molecule libraries largely determines the success of the HTS approach. Natural products, the small molecules produced by many living organisms, contain truly remarkable levels of molecular diversity and have a long history of success in discovering new drugs. But in spite of this history, they play only a minimal role in today's HTS world. This project describes two approaches, which minimize some of the liabilities of traditional natural products discovery techniques that could lead to an important small molecule stream for HTS.